Molecular diagnosis is a leading-edge technology in modern biological analysis and detection of specific nucleic acid sequence is an important part of biological analysis. Conventional molecular identification methods, such as fluorescent quantitative PCR (Qi Y. Appl Environ Microbiol, 2001, 67 (8): 3720-3727), RFLP (restriction fragment length polymorphism, S R Klee. J. Applied Microbiology. 2006, 100, 1364-5072), gene chip and sequencing, are complex and require large, expensive equipment and long time. They are very inaccessible for quick detection of some pathogenic micro-organisms (biological weapons) like Bacillus anthracis, Therefore, the development of an alternative convenient, cheap, efficient, equipment-free analyzing means for the healthcare site use has very important practical significance.
Gold nano-particle (AuNP) is a colloidal bio-molecular marker with a diameter of 1-100 nm, enjoying advantages as good stability, small size effect, surface effect, quantum effects, optical effects, and unique biological affinity, widely used in the optics, electrics, and imaging (Horisberger M. Scanning Electron Microscopy II, 1981: 9-31). AuNP has a high extinction coefficient and its particle with a diameter of 13 nm has a sharp absorption peak at about 520 nm wavelength. Its self-assembled aggregation can significantly alter the absorption peak, and the surface plasmon resonance (SPR) can lead to a color change. Meanwhile, AuNP has a wide range of biological affinity coupling the DNA molecules and participating the self-assembly process, ultimately affecting physical properties of the AuNP solution (or colloidal gold solution), such as color and absorbance. Mirkin found self-assembly aggregation of the AuNP-DNA particles and color change in the colloidal solution caused by specific hybridization of DNA, first applying the AuNP to the biological detection (Mirkin C A. Nature, 1996, 382 (6592): 607-609). AuNP can couple thiol modified short chain DNA forming a detection probe, and when complementary same-length DNA exists in the solution, orderly and reversible agglomeration reaction occurs, developing two-dimensional, three-dimensional agglomeration structure in mesh and causing color change from ruby red to purple blue (Elghanian R. Science, 1997, 277 (22): 1078-1081). This naked-eye visible effect of AuNp forebodes a quick, easy way in biodetection. ssDNA (single stranded DNA) and dsDNA (double stranded DNA) are different in absorption of the AuNP, with the former to the negatively charged surface of AuNP making it in a stable state free from electrolyte salt ions' aggregation effect (Li H. PNAS, 2004, 101 (39): 14036-14039), Li accordingly designed nucleic acid hybridization colorimetric detection on the basis of the AuNP aggregation reaction. A conventional PCR is conducted against the target genes of the cardiac arrhythmias obtaining a double-stranded product. Then colloidal gold solution and the probes are added, after denaturation annealing, the colloidal gold solution turns from ruby red to purple blue if specific target nucleic acid has been amplified, if not, ruby red persists (Li H J, Am Chem Soc., 216: 10958-10961, 2004). This new idea for nucleic acid detection is a non-crosslinked manner not requiring covalent modification of the AuNP and probe. However, in the actual operation, the ratio of the probe and the PCR product is uneasy to control, requiring large amount of pretest or purification and quantitative determination of the analytes of unknown concentration, so this method is unacceptable for application.
Recently, the combination of AuNP's SPR effect and the PCR produces a Nano-PCR detection method. Cai used an oligonucleotide probe coupled AuNP to supersede the conventional primer, amplifying exon sequence of HIV gp140 using PCR followed by colorimetric detection by the naked eyes. Although it is quick and easy (Miao Cai. Nano Res. 2010, 3: 557-563), the color change is not significant, limiting its applicability.
The above-mentioned calls for a simple and easy nucleic acid detection method with obvious color change.